Protocol for the thermodynamic analysis of some proteins using an H/D exchange- and mass spectrometry-based technique

Anal Chem. 2005 Jan 15;77(2):693-7. doi: 10.1021/ac048967z.

Abstract

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a new H/D exchange- and mass spectrometry-based technique for the measurement of protein folding free energies (i.e., DeltaG values) and protein folding m values (i.e., deltaDeltaG/delta[denaturant]). Robust protocols for the acquisition and analysis of SUPREX data have been established and shown to be useful for the analysis of a number of different protein systems. Here we report on the SUPREX behavior of a special class of proteins that are not amenable to conventional SUPREX analyses using previously established protocols. This class of proteins includes protein systems that require an extended time to reach a folding/unfolding equilibrium in chemical denaturant-induced equilibrium unfolding experiments. As part of this work we use ubiquitin as a model system to highlight the complications that can arise in the conventional SUPREX analysis of such protein systems, and we describe a modified SUPREX protocol that can be used to eliminate these complications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Deuterium Exchange Measurement / methods*
  • Protein Denaturation
  • Protein Folding
  • Proteins / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Thermodynamics
  • Time
  • Ubiquitin / chemistry*

Substances

  • Proteins
  • Ubiquitin